We have recently isolated nitrogen-fix nitrogen-fixing bacteria that have molybdenum-independent nitrogenases from natural environments (waste water treatment plants, ponds, streams, and salt marshes) using molybdenum (Mo)- and nitrogen-free enrichment media. We hypothesize that non-molybdenum nitrogenases are important under natural conditions even in the presence of ordinarily sufficient molybdenum because micro environments devoid of Mo may be created by removal of Mo by the microorganisms themselves and by other organisms such as plants. We propose to extend our survey to rhizospheres and rhizoplanes of C4 plants such as sugarcane, corn, and spartina grass (in salt marshes) where conditions should favor creation of Mo-deficient micro niches. Nitrogen-fixing bacteria isolated the rhizosphere and rhizoplane of plants will be phylogenetically characterized using 16S rRNA sequences and the presence or absence of alternative nitrogenase systems will be determined using PCR primers specific for amplification of genes specific for each nitrogenase system. Representive strains will be further characterized with respect to nitrogen fixation capabilities and ability to colonize the rhizosphere and rhizoplane of plants. To our knowledge the use of Mo-deficient enrichment media have not been used to isolate diazotrophs directly from these environments. Thus, it can be expected that diazotrophs with novel properties will be identified during this study. The intent of the proposed study is to further our understanding of why certain diazotrophic organisms have Mo-independent nitrogenases and the role that these nitrogenases might play in the global nitrogen cycle.